Chicken leukemia inhibitory factor (LIF) and GNNE thereof

ABSTRACT

The present invention is to provide a chicken LIF gene. Based on this genetic information, LIF protein derived from the chicken can be stably supplied and it solves the problems of the creation of transgenic chickens in the past. In addition, the present invention provides not only transgenic chickens for testing purposes but also supplies the first practical transgenic stock animals. The present invention relates to leukemia inhibitory factor (LIF) shown in sequence No. 2, and the gene that encodes thereof shown in sequence No. 1, and a manufacturing method of chicken LIF. In addition, the present invention pertains to a differential preventer of the chicken differentiable cell, and a method of chicken differential prevention and a culturing method for the chicken differentiable cell using thereof, and a medium comprising thereof.

RELATED APPLICATIONS

This application is a divisional of patent application Ser. No.11/102,749, filed on Apr. 11, 2005, which is a divisional of patentapplication Ser. No. 10/061,375, filed on Feb. 4, 2002, the entirecontents of which are incorporated herein by reference.

FIELD OF TECHNOLOGY

The present invention relates to a chicken leukemia inhibitory factor(hereinafter referred to as “LIF”) and the gene thereof, as well as itsmanufacturing method. The chicken LIF of the present invention has adifferentiation prevention function for a chicken differentiable cellsuch as a chicken's embryonic stem cell or embryonic reproductive cell,and is effective as an anti-differentiator while the chickendifferentiable cell is being cultured.

The present invention also relates to a culturing method for a chickendifferentiable cell using a chicken LIF, a culture medium, and acreation method for a transgenic chicken using said method, and atransgenic chicken created with said method.

BACKGROUND OF THE INVENTION

Recently, the creation of transgenic animals has brought significantprogress in a wide range of areas including medicine and biology. Thecreation of animals that are introduced with specific genes orknocked-out specific genes has become an important tool to analyze thefunctions of these genes. There have been two types of creation methodsfor transgenic animals: the transgenic method in which a foreign gene isdirectly introduced to a fertilized egg, and the gene introductionmethod that is carried out via an embryonic stem cell (hereinafterreferred to as an ES cell). Currently, the introduction of a gene to theES cell has been widely used because the target gene can be introducedor there is a knock-out from a designated genome location using the genetargeting method.

As described above, the creation of transgenic animals using the ES cellby the gene targeting method has been widely carried out. In thismethod, the inhibition of the differentiation during the engineering ofthe gene is the significant issue. To culture an ES cell, thepreparation of a feeder cell or the use of an LIF as a factor to inhibitthe differentiation is required. The LIF is a type of cytokine thatbelongs to the IL-6 family. It is an essential material to maintain thecell in an undifferentiated state (Smith A., et al., Nature, 336,688-690 (1988)).

A mouse LIF is an essential factor to create a transgenic mouse throughthe gene targeting method, and the recombinant LIF derived from themouse has been commercialized. The culturing of a mouse ES cell has beenrealized using this factor only, however, the LIF has not beenestablished for other animals and the creation of a transgenic animalusing the gene targeting method has been difficult.

The creation of transgenic chickens has been attempted because its egghas a high productivity of proteins. The fertilized egg of the chickenis relatively easy to handle so that the introduction of genes has fewproblems. However, in order to introduce a knock-out of the gene to orfrom the designated location, the creation of a transgenic chicken usingthe gene targeting method is necessary.

Chickens and mice have different development patterns so that the EScell cannot be used. However, it is possible to separate and culture acell equivalent to the ES cell from the developing embryo of a chicken.Chickens have blastodermic cells and primordial reproductive cells thatare equivalent to a mouse ES cell. Blastodermic cells are developingreproductive cells that are equivalent to the mouse ES cell. Theprimordial reproductive cell is a cell that is going to bedifferentiated to be a reproductive cell, and the development of a stockcell that can be externally cultured has advanced. This stock cell iscalled an embryonic germ cell. The issue is how to perform the culturingwhile maintaining the undifferentiated state for either cell. In anyevent, currently no chicken LIF has been found so that a medium that isa combination of another mammal LIF and another cytokine has been used.For example, a study has been carried out to establish a culturingsystem for chicken ES cells and embryonic reproductive cells using arecombinant LIF derived from a mouse. However, its effect is low andcurrently no culturing system has been established.

The chicken embryo is easy to obtain and the operation for the embryo issimple so that it has been widely used as a study material inembryology. In addition, the establishment of a production technologyfor transgenic chickens has been desired because chickens are not onlyused for their meat but also they have a high productivity of protein intheir eggs, and are industrially important. However, a culturing systemfor the chicken ES cell and embryonic reproductive cell has not beenestablished so that we have not reached the level where an artificialmodification of the gene can be easily carried out (at the level of thetransgenic mouse). Thus, the gene/protein cloning of chicken LIF, whichis an essential factor for the culturing system is being quickened.Nevertheless, chickens are significantly different from other mammalsand even the fact as to whether an LIF exists or not has not beendetermined. As described above, the manufacturing of chicken LIF proteinhas been desired in order to create transgenic chickens.

SUMMARY OF THE INVENTION

The present invention has taken advantage of state-of-the-art molecularbiological techniques in order to solve these problems and to provide agene for a chicken LIF. If a stable supply of the LIF protein derivedfrom chickens is possible based on this genetic information, the problemof the creation of a transgenic chicken in the past can be resolved.

In addition, the present invention mass produces the recombinant LIFprotein based on the genetic information of the present invention, and asupply thereof as the medium reagent for the chicken ES cell andembryonic reproductive cell in order to create a transgenic chicken.Consequently, it allows the providing of not only transgenic chickensfor test purposes but also the first practical transgenic stock animals.

The present invention relates to a chicken LIF, and the gene thatencodes thereof, and a manufacturing method for chicken LIF.

The present invention also relates to a differential preventer of achicken differentiable cells comprising the chicken LIF, thedifferentiation prevention method of the chicken using thereof, aculturing method for the chicken differentiable cells, and the mediumcomprising thereof.

The present invention further relates to a method of creating transgenicchickens in which said differentiable cell is cultured in the presenceof a chicken LIF, and the transgenic chicken that is created using saidmethod.

The inventors of the present invention stimulated chicken monocytic cellstock (IN24) with a lipopolysaccharide (LPS), and using the subtractionmethod, the gene that excessively developed due to the LPS stimulation,was cloned. Additionally, a clone that is homological (approximately30%) to the LIF derived from a mammal was obtained among these clones.

The cDNA base sequence of the obtained clone is shown in sequence No. 1in the sequence table. This gene is comprised of 789 bases and it isassumed that it encodes the protein comprising 211 amino acids having aninterpretation area of 75th atg to 708th tga.

The sequence of chicken LIF that is comprised of the assumed 211 aminoacids is shown in sequence No. 2 in the sequence table. The one lettercode of the amino acid of this amino acid sequence is shown as follows.

MRLIPAGVVP FVALLLLQRR PVSGRALLGT  30 SSACPTNGLC RANVLEQTRR QVALLNATAQ 60 DLFSLYLKCQ GEPFSSESDR LCSPSGIFFP  90 PFHVNRTTER KEVMVAMYKLFAFLNASLGN 120 ITRDQEELNP MAKELLNRLH NTTKTTRGLI 150 SNLTCLLCKHYNIFQVDVSY GESSKDKSAF 180 KKKQQGCQVL RKYVQVIAQA ARVLLPHLSP 210 A 211

In addition, the base sequence and amino acid (one letter code) of theobtained chicken LIF gene is shown in FIG. 1.

A comparison of the amino acid sequence assumed from the base sequenceof the chicken LIF of the present invention with the amino acid sequenceof the human LIF and mouse LIF is shown in FIG. 2. The black triangle inFIG. 2 shows the location of the cysteine and the white triangle showsthe location where a saccharide can be added.

The amino acid sequence assumed from the base sequence of the chickenLIF of the present invention is homologous for less than 40% with themouse LIF. Only a partial effect has been recognized with the culturesystem of the chicken ES cell and embryonic reproductive cell using therecombinant mouse LIF. This supports the fact that the mouse LIF andchicken LIF have a low homologue. However, the location of the cysteine,which is an important amino acid that determines the structure ofprotein, was completely identical among the chicken, human and mouse.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a chicken amino acid sequence and the DNA base sequence ofthe present invention.

FIG. 2 shows a comparison of the chicken LIF amino acid sequence withthe human LIF and mouse LIF thereof.

FIG. 3 is a photo in place of a drawing that shows a selected clone fromthe clones obtained by the subtraction method.

FIG. 4 shows the location of the primer and its sequence when the RACEmethod is applied to the clones obtained by the subtraction method.

FIG. 5 shows the schematic structure of a cDNA base sequence of achicken LIF of the present invention. The arrow shows the location ofthe primer of the RT-PCR method that was carried out in order to confirmthe sequence.

FIG. 6 is a photo in place of a drawing that shows the results of a 502bp fragment obtained by the RT-PCR method.

PREFERRED EMBODIMENTS OF THE INVENTION

The chicken LIF of the present invention is not limited to the onehaving the amino acid sequence shown in sequence No. 2 in the sequencetable. As long as it is LIF active for a chicken, a portion of the aminoacid shown in sequence No. 2 can be deleted, or substituted with anotheramino acid, and/or added with another amino acid.

There is no particular limitation on the number of amino acids fordeletion, substitution or addition, however it is preferable for it tobe half or less, which is 1 to 100 or 1 to 50 pieces.

The chicken LIF gene of the present invention is not limited to those ofthem that have the base sequence shown in sequence No. 1 of the sequencetable. As long as it encodes the protein that is LIF active for thechicken as described above, a portion of the amino acid shown insequence No. 1 can be deleted, or substituted with another amino acid,and/or added with another amino acid. In addition, the chicken LIF geneof the present invention can be a double strand DNA or single strand DNAor RNA. Those of them that have a supplemental base for said chicken LIFof the present invention are included in the chicken LIF gene of thepresent invention. Furthermore, the chicken LIF gene of the presentinvention includes a base sequence that can be hybridized understringent conditions.

In addition, the present invention provides a fragment of a chicken LIFgene of the present invention. The fragment of the chicken LIF gene ofthe present invention can be used as a probe to detect, identify orquantify the chicken LIF gene of the present invention, and as a primerin order to obtain the chicken LIF gene of the present invention.

As a fragment of the chicken LIF gene of the present invention,approximately 5 to 50 bases, 5 to 30 bases or 10 to 30 bases of thesequence from an arbitrary location can be used.

The manufacturing method of the chicken LIF of the present invention canbe carried out using a well-known method using said chicken LIF gene ofthe present invention. For example, a gene that has the base sequenceshown in sequence No. 1 is combined into a manifestation vector and aphenotypic transformation of the host cell is carried out. Additionally,it can be manufactured through the culturing of the phenotypicallytransformed host cell. A prokarytoic cell such as E. coli or aneukaryotic cell such as a cell from yeast or a mouse can be used as thehost cell.

Based on the chicken LIF gene of the present invention, a recombinantchicken LIF can be created using a prokarytoic cell or a eukaryoticcell, and consequently, the mass production of the chicken LIF necessaryto create a transgenic chicken can be realized.

Examples of the chicken differentiable cell of the present inventionare, chicken ES cells and embryonic reproductive cells.

When a chicken differentiable cell is cultured, the chicken LIF of thepresent invention can prevent the differentiation of said cell by beingadded in the culture system. Therefore the chicken LIF of the presentinvention can be used as the differentiation preventer of the chickendifferentiable cell. Purified chicken LIF of the present invention canbe individually used as the differentiation preventer of the presentinvention or it can be used in a mix with a carrier. More over, a mediumto culture the chicken differentiable cells can be created by mixing theLIF of the present invention with the medium in advance.

Furthermore, the present invention provides a culturing method for thechicken differentiable cells using the LIF of the present invention. Itprovides a culturing method for a chicken differentiable cells by addingsaid differentiation preventer of the present invention into the culturesystem, or using said medium of the invention.

In addition, the present invention provides a creation method for atransgenic chicken and a transgenic chicken created using the methodhereof. An example of the desirable creation method of the transgenicchicken of the present invention is such that the differentiable cellssuch as the chicken ES cells and embryonic reproductive cells withmodified genetic information are modified and cultured thereof:preferably by introducing a foreign gene that can be developed at aspecific genome location through a gene targeting method; or through amodification in order to inhibit the development of a specific gene atspecific genome locations, for instance, an introduction of a foreigngene to destroy an Exon area such as a promoter area or a modificationof the gene to modify or add encoded amino acid. When chicken ES cellsor embryonic reproductive cells are used as the differentiable cells,normally, a chimera chicken is obtained. Additionally, the nextgeneration thereof becomes a transgenic chicken in which the target geneis introduced or knocked out.

The recombinant chicken LIF created based on the chicken LIF gene of thepresent invention is a discovery with hope in order to provide anestablished method for the creation of transgenic chickens. The use ofthis allows the maintenance of the subculture of the chicken ES cellsand embryonic reproductive cells in an undifferentiated state, which hasbeen impossible in the past, and also allows the in-vitro selection ofthe introduced gene, which has been difficult for chickens. In otherwords, all the problems regarding the creation of a transgenic chickenhave been resolved with the present invention.

In addition, when the culture system of these cells is established, thecreation of a transgenic chicken will become even simpler than a mouse,allowing the poultry industry to produce (produced as eggs) utilitymaterials (medical drugs, reagents for clinical tests, etc.).

Embodiments

The present invention is described in detail using embodiments asfollows, however, the present invention is not limited to theseembodiments.

Embodiment 1 Gene Cloning of the Chicken LIF

mRNA was purified from chicken monocytic cell stock (IN24) stimulatedwith a lipopolysaccharide (LPS) and not stimulated separately, and thendouble strand cDNA was synthesized.

Using the cDNA obtained from these two types of cells, a subtractionmethod was carried out using the Clontech PCR-select cDNA subtractionkit. The cDNA obtained was cloned to the pGEM-T vector. These cloneswere phenotypically transformed to JM 109 E-coli and cultured on an agarmedium and then the developed colony was selected using the PCR method.It was selected such that the PCR was carried out using the pUC-M 13forward primer and reverse primer in the pGEM-T vector base sequence andonly those clones with cDNA inserted in the vector were selected. Theresults are shown in FIG. 3 as a photo in place of a drawing.

Finally, 122 clones were selected and the plasmid of these clones wascreated. Then using an auto-sequencer, the base sequence of each clonewas determined.

Each of the determined base sequences were checked against a database,and then it was found that one of the clones is a clone that ishomologous with a mammalian (cows, pigs, etc.) derived LIF.

As described above, we determined that the obtained clone is a part ofthe gene (259 bp) that encodes the chicken LIF. Based on this basesequence of the clone, the cloning of the complete chicken LIF wascarried out by the RACE method using the Clontech smart-RACE kit.

Based on the obtained chicken LIF gene base sequence, at the locationshown in FIG. 4, primers for 3′ and 5′ were prepared and a PCR wascarried out between the adaptor sequences. The top line of FIG. 4 is aschematic view of the 259 bp sequence obtained as above, and the bottomline shows its specific sequence.

As a result, 789 bp was determined including the entire translated area.

The determined base sequence was confirmed from the fragment obtainedthrough the RT-PCR method using the new primer shown in FIG. 5. Thesquare-framed portion in FIG. 5 shows the translated area and theshadowed portion shows the 259 bp portion obtained through saidsubtraction method. The arrow in the bottom line in FIG. 5 shows theprimer portion of the RT-PCR.

The results of electrophoresis of the 502 bp fragment amplified by theRT-PCR method are shown in FIG. 6 as a photo in place of a drawing. Thelane M indicates the marker and the black triangle shows the location ofthe 502 bp fragment.

The base sequence and amino acid (one letter code) of the obtainedchicken LIF gene is shown in FIG. 1. Its base sequence is shown insequence No. 1 and the amino acid sequence is shown in sequence No. 2.

The present invention is to provide a chicken LIF and the gene thereofthat is necessary to create a chicken with introduced foreign genes(transgenic chicken) for the first time. The present invention is on thefrontier of the creation of a chicken with introduced foreign genes(transgenic chicken) and in addition, it is expected to lead to the massproduction of utility materials such as medical drugs. So far, theprogress of studies and the practical use of the creation of atransgenic chicken have been extremely delayed because a chicken LIF hasbeen unknown, however, the providing of the chicken LIF of the presentinvention allows practical use to the same extent as that for a mouse.

It is easy to obtain the embryo of a chicken and it has goodoperability. The chicken LIF of the present invention allows theestablishment of a culturing system of chicken ES cells, allowing thecreation of transgenic chickens, which is even easier than that for amouse. This is not just at the laboratory level but also it allows theapplication to breeding in the poultry industry. In addition, becausethe chicken egg has a high productivity of protein, it allows thepractical manufacturing of utility materials (medical drugs, clinicaltest reagents, etc.). The present invention opens up the road for thefirst time to a supply of practical level transgenic stock animals.

The disclosure of Japanese Patent Application No. 2001-171993 filed Jun.7, 2001 including specification, drawings and claims are hereinincorporated by reference in their entirety.

Although only some exemplary embodiments of this invention have beendescribed in detail above, those skilled in the art will readilyappreciated that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention.

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